An important aspect in the fields of medical diagnostics and biomedical research involves detection, identification, quantification, and characterization of various cells and biomolecules of interest through testing of biological samples such as blood, spinal fluid, cell culture and urine. Healthcare providers and biomedical researchers routinely analyze such biological samples for the microscopic presence and concentrations of cells and biomolecules.
Fluorescence microscopy, fluorescent plate reader, and flow cytometry are traditionally used for fluorescence detection with cell-based assays. These methods utilize glass slides, microtiter plates, and flow chamber to perform fluorescence analysis. These fluorescence detection methods, however, often incorporate expensive excitation light sources such as lasers or arc lamps for high intensity excitation. Typically, there is an excitation light source and a detection probe with an emission filter to pick up specific fluorescent signals. In an instrument such as fluorescent microscope, for example, a dichroic filter is required to reflect the light from the top normal (i.e., at a right angle) to the sample chamber. The emitted fluorescence is then picked up by passing through the dichroic filter into a detector such as a camera, spectrometer, etc. These fluorescence detection methods are also not used to directly measure sample concentration, since they do not measure samples in a specific fluid volume.
An example of a previous fluorescent cell counting technology is one that utilizes a filter cube (such as those provided by Omega Optical) in an assembly that includes optics, a camera, and a sample holder. Such as assembly may provide sufficient fluorescent images of cells and other biosamples for a number of applications, and it provides a simple and efficient method to generate fluorescent images of biological samples. This technology utilizes a fixed chamber volume, which is used to directly measure sample concentration, while analyzing fluorescence intensity. It, however, lacks the sensitivity for low fluorescent signal detection and imaging. One issue with such a system, for example, is that the excitation light may leak out into the emission filter in the filter cube. Another issue is that the filter cube format generally is inflexible in color selection. Only one specific filter cube and one LED could be used for one color. There is not much space in the instrument to incorporate other colors, making it difficult to allow multi-color applications.
Therefore, a long-felt need exists for cell counting systems and methods that provide capabilities for detection and imaging of low fluorescent signals such as that of surface markers on various types of cells.